Cellular response mechanisms to stress are fundamentally important to the human immune system. Stress responses represent carefully devised cellular defense mechanisms which were developed at an early point during evolution; evidenced by the fact that biomolecules implicated in stress response exhibit remarkable similarity across the animal kingdom. Welch, W. J., et al., The Stress Response and the Immune System, Inflamnmation: Basic Principles and Clinical Correlates, Raven Press, Gallin, J. I., et al., Eds., Second Edition, 41:841 (1992).
Lymphocyte activation, homing, resistance to target cell lysis, tumor antigenicity, regulation of proto-oncogene transcription, and immune surveillance are examples of immunologic functions that appear to be mediated or modulated by stress activated signal transduction molecules. Siegelman, M., et al., Science, 231:823 (1986); Kusher, D. I., et al., J. Immunol., 145:2925 (1990); Ullrich, S. J., et al., PNAS, 83:3121 (1986); Colotta, F., et al., Biochem. Biophys. Res. Commun., 168:1013 (1990); Haire, R. N., et al., J. Cell Biol, 106:883 (1988); Born, W., et al., Immunol. T., 11:40 (1990). The number of preactivated and MHC class II-restricted autoreactive T-lymphocytes in peripheral blood of patients with rheumatoid arhritis, for example, dramatically increases relative to the levels in healthy individuals. Similarly, peripheral blood T-lymphocytes from patients with inflammatory arthritis proliferate strongly in the absence of exogenous antigen or mitogen. Welch, W. J., et al., The Stress Response and the Immune System, Inflammation: Basic Principles and Clinical Correlates, Raven Press, Gallin, J. I., et al., Eds., Second Edition, Chapter 41, 841 (1992). Moreover, synovitis has been shown to result in the generation of oxygen-derived free radicals that act to perpetuate tissue damage. Blake, D. R., et al., Hypoxic-Reperfusion Injury in the Inflamed Human Joint, Lancet, 2:2889 (1989).
The control of hematopoiesis is a highly regulated process that responds to a number of physiological stimuli in the human body. Differentiation, proliferation, growth arrest, or apoptosis of blood cells depends on the presence of appropriate cytokines and their receptors, as well as the corresponding cellular signal transduction cascades. Hu, Mickey C.-T., et al., Genes & Development, 10:2251(1996). Generation of mature leukocytes, for instance, is a highly regulated process which responds to various environmental and physiological stimuli. Cytokines cause cell proliferation, differentiation or elimination, each of these processes being dependent on the presence of appropriate cytokine receptors and the corresponding signal transduction elements. Moreover, the stimulation of quiescent B- and T-lymphocytes occur via antigen receptors which exhibit remarkable homology to cytokine receptors. Grunicke, Hans H., Signal Transdiuction Mechanisms in Cancer, Springer-Verlag (1995). See also, Suchard, S. J., et al., Mitogen-Activated Protein Kinase Activation During IgG-Dependent Phagocytosis in Human Neutrophils, J. Immunol., 158:4961 (1997).
Distinct signaling cassettes, each containing a central cascade of kinases, respond to a variety of positive and negative extracellular stimuli, lead to changes in transcription factor activity and posttranslational protein modifications in mammalian cells. Kiefer, F., et al., EMBO, Vol. 5, 24:7013 (1996). One such protein kinase cascade, known as the mitogen-activated protein kinase (MAPK) cascade, is activated as an early event in the response of leukocytes to various stimuli. Stimulation of this pathway has been observed during growth factor-induced DNA synthesis, differentiation, secretion, and metabolism. The MAPK pathway has a critical role in the transduction of receptor-generated signals from the membrane to the cytoplasm and nucleus. Graves, J. D., et al., Protein Serine/Threonine Kinases of the MAPK Cascade, Annals New York Academy of Sciences, 766:320 (1995). It has been established that sustained activation of the MAPK cascade is not only required, but it is sufficient to trigger the proliferation of some cells and the differentiation of others. Cohen, P., Dissection of Protein Kinase Cascades That Mediate Cellular Response to Cytokines and Cellular Stress, Advances in Pharmacology, Academic Press, Hidaka, H., et al., Eds., Vol. 36, 15 (1996); Marshall, C. J., Cell, 80:179 (1995). Several interdependent biochemical pathways are activated following either stimulation of resting T-lymphocytes through the antigen receptor or stimulation of activated T-lymphocytes through the interleukin-2 (IL-2) receptor. Many of the events that occur after the engagement of either of these receptors are qualitatively similar, such as the activation of mitogen-activated protein kinase (MAPK) pathways and preexisting transcription factors, leading to the expression of specific growth-associated genes. Symmetry of the Activation of Cyclin-dependent Kinaes in Mitogen and Growth Factor-stimulated T Lymphocytes, Jaime F. Modiano, et al., Annals New York Academy of Sciences, 766:134 (1995).
Recent evidence suggests that cellular response to stress is controlled primarily through events occurring at the plasma membrane, overlapping significantly with those important in initiating mitogenic responses. Exposure of cells to biological, chemical, or physical stress agents evokes a series of events leading to the activation of a wide group of genes including transcription factors as well as other gene products that are also rapidly and highly induced in response to mitogenic stimulation. The mitogen-activated protein kinase (MAPK) pathway has been shown to be essential for the mitogenic reponse in many systems. See, e.g., Qin, Y. et al., J.Cancer Res.Clin.Oncol., 120:519 (1994). Moreover, due to the fact that most oncogenes encode growth factors, growth factor receptors, or elements of the intracellular postreceptor signal-transmission machinery, it is becoming increasingly apparent that growth factor signal transduction pathways are subject to an elaborate network of positive and negative cross-regulatory inputs from other transformation-related pathways. Grunicke, Hans H., Signal Transduction Mechanisms in Cancer, Springer-Verlag (1995). The Hierarchical organization of the MAPK cascade makes integral protein kinase members particularly good targets for such "cross-talk". Protein Serine/Threonine Kinases of the MAPK Cascade, J. D. Graves, et al., Annals New York Academy of Sciences, 766:320 (1995).
Initial triggers for inflammation include physical and chemical agents, bacterial and viral infections, as well as exposure to antigens, superantigens or allergens, all of which have the potential to generate Reactive Oxygen Species (ROS) and to thereby activate second messenger signal transduction molecules. Storz, G., et al., Transcriptional Regulators of Oxidative Stress-Inducible Genes in Prokaryotes and Eukaryote, in: Stress-Inducible Cellular Responses, Feige, U., et al., Eds., Birkhauser Verlag (1996). Reactive oxygen radicals, via damage to many cellular components including DNA, can cause cell death or, if less severe, cell cycle arrest at growth-phase checkpoints. Stress damage not only activates checkpoint controls but also activates protein kinases, including the stress activated protein kinases (SAPKs), c-Raf-1 and ERKs, which are integral components of cytoplasmic signal transduction (MAPK) cascades. Pombo, C. M., et al., EMBO, Vol. 15, 17:4537 (1996); Russo, T., et al., J.Biol. Chem., 270:29386 (1995). Considering, inter alia, that stress has also been implicated in oxidant injury, atherosclerosis, neurogenerative processes, and aging, elucidation of the components of mammalian stress-induced pathways should provide more specific targets that can be exploited therapeutically. N. J. Holbrook, et al., Stress Inducible Cellular Responses, 273, U. Feige, et al., Eds., Birkhauser Verlag (1996).
Evidence has demonstrated that mitogen-activated protein kinase (MAPK) and stress activated protein kinase (SAPK) signal transduction pathways are responsible for triggering biological effects across a wide variety of pathophysiological conditions including conditions manifested by dysfunctional leukocytes, T-lymphocytes, acute and chronic inflammatory disease, auto-immune disorders, rheumatoid arthritis, osteoarthritis, transplant rejection, macrophage regulation, endothelial cell regulation, angiogenesis, atherosclerosis, fibroblasts regulation, pathological fibrosis, asthma, allergic response, ARDS, atheroma, osteoarthritis, heart failure, cancer, diabetes, obeisity, cachexia, Alzheimers disease, sepsis, and neurodegeneration. As MAP kinases play a central role in signaling events which mediate cellular response to stress, their inactivation is key to the attenuation of the response. N. J. Holbrook, et al., Stress-Inducible Cellular Responses, 273, Feige, U., et al., Eds., Birkhauser Verlag (1996).
Despite major efforts to develop new therapeutic approaches Adult Respiratory Distress Syndrome (ARDS) (acute pulmonary inflammation characterized by the massive generation of Reactive Oxygen Species (ROS) within the lung), for example, remains lethal for about 50% of affected patients. Polla. B.S., et al., Stress Proteins in Inflammation, in: Stress Inducible Cellular Responses, Feige, U., et al. Eds., Birkhauser Verlag (1996). Moreover, the chronic inflammatory disease, rheumatoid arthritis, for instance, is believed to be mediated by activated T-lymphocytes that infiltrate the synovial membrane and initiate a series of inflammatory processes. Panayi, G. S., et al., The Importance of the T-Cell in Initiating and Maintaining the Chronic Synovitis of Rheumatoid Arthritis, Arthritis Rheum, 35:729 (1992). Accumulating evidence also indicates that the autoimmune disease multiple sclerosis (MS) is mediated by autoreactive T-lymphocytes. Stinissen, P., et al., Crit. Rev. Immunol., 17(1):33 (1997). Autoreactive T-lymphocytes have been demonstrated to undergo in vivo activation and clonal expansion in patients with MS. Zhang, J., et al., J. Mol. Med., 74(11):653 (1996). In diabetes mellitus, autoreactive T-lymphocytes systematically destroy pancreatic islet cells such that they prove incapable of producing insulin. Another propelling recent development in the implication of overactive T-cells is the recognition that a particular subset of T-lymphocytes appear to be a major culprit in asthma and other allergic diseases, by responding with undue vigor to apparently harmless invaders (rates of asthma per capita in the developing world have increased dramatically in the last several decades; doubling in the U.S. since 1980). New Clues to Asthma Therapies: Vogel, G., Science, 276:1643 (1997).
Recently, much progress has been made in defining the signal transduction pathways mediating the cellular response to stress. Pombo, C. M., et al., for instance, report the cloning and characterization of a human Ste20-like oxidant stress response kinase, SOK- 1.The kinase is positively regulated by phohsphorylation and negatively regulated by its C-terminal non-catalytic region. Reported data suggests SOK-1 transduces signals in response to oxidative and environmental stress. EMBO, Vol. 15, 17:4537 (1996). Moreover, Schinkmann, K., et al., recently reported the cloning and characterization of the human STE-20-like kinase, mst-3. The mst-3 transcript is reported to be ubiquitously expressed. Mst-1 is furthermore reported to be positively regulated by autophosphorylation. J. Biol. Chem., 272(45):28695 (1997).Other stress-activated protein kinase (SAPK), members of the MAPK family, have been shown to be activated in situ by inflammatory stimuli, including tumor-necrosis factor (TNF) and interleukin-1. Kyriakis, J. M., et al., Nature, 369:156 (1994); Derijard, B., et al., Cell, 76:025 (1994); Sanchez, I., et al., Nature, 372:794 (1994). See also, Kiefer, F., et al., EMBO, Vol. 5, 24:7013 (1996); Creasy, C. L., et al., J. Biol. Chem., 271: No. 35, 21049 (1996)); Creasy, C. L., et al., Gene, 167:303 (1995)); Manser, E., et al., Nature, 367:40 (1994); Hu, Mickey C.-T., et al., Genes & Development, 10:2251(1996); Katz, P., et al., J. Biol. Chem., (1994)); Pombo, C. M., et al., Nature, 377:750 (1995).
Integral members of cellular signaling pathways as targets for therapeutic development, for example, have been the subject to several reviews. See, e.g., Levitzki, A., Signal-Transduction Therapy: A Novel Approach to Disease Management, Eur. J. Biochem, 226:1 (1994); Powis G., The Potentialfor Molecular Oncology to Define New Drug Targets, in: New Molecular Targets for Cancer Chemotherapy, Workman, P., Kerr D. J., eds., CRC Press, Boca Raton FL (1994). As a result of the efforts of numerous laboratories, an impressive list of remarkably specific inhibitors of kinases, for instance, has become available. See, e.g., Levitzki, A., Tyrphostins: Tyrosine Kinase Blockers as Novel Antiproliferative Agents and Dissectors of Signal Transduction, FASEB; 6:3275 (1992); Workman P., et al., Discovery and Design of Inhibitors of Oncogenic Tyrosine Kinases, in: New Approaches in Cancer Pharmacology: Drug Design and Development, Springer, Berlin 55 (1994).
A novel class of pyridinyl imidazoles, CSAIDS [SKB], for instance, have been developed, that inhibit the production of the cytokines interleukin-1 (IL- 1) and tumor necrosis factor (TNF-.alpha.) in monocytes. The drug has been demonstrated to bind specifically to one protein in monocytes, termed CSBP (CSAID-binding protein), which has been isolated, cloned, and sequenced and demonstrated as a MAPK homolog. Lee, J. C., et al., Differential Effects of the BicyclicItnidazoles on Cytokine Synthesis in Human Monocytes and Endothelial Cells, Agents Actions, 41: C191 (1994); A Protein Kinase Involved in the Regulation of Inflammatory Cytokine Biosynthesis, Nature, 372:739 (1994). Moreover, as demonstrated by the identification of rapamycin as a specific inhibitor of the activation of p70 S6 kinase and the identification of compounds that inhibit the EGF receptor protein kinase very potently and that block the activation of MAP kinase kinase have demonstrated that specific inhibitors of protein kinases can indeed be developed. Alessi, D., et al., A Specific Inhibitor of the Activation of MAP Kinase Kinase-1 in vitro and in vivo, J. Biol. Chem., 279:27489 (1995); Fry, D., et al., A Specific Inhibitor of the Epidermal Growth Factor Receptor Tyrosine Kinase, Science, 265:806 (1994); Cohen, P., Dissection of Protein Kinase Cascades That Mediate Cellular Response to Cytokines and Cellular Stress, Intracellular Signal Transduction, Advances in Pharmacology, Hidaka, H., et al., Eds., Academic Press, 36:17 (1996).
Compounds which are able to modulate the activity of specific signal transduction molecules integral to specific intracellular pathways are expected to have significant potential for the ability to control or attenuate downstream physiological responses. Unfortunately, in spite of the introduction of numerous new drugs during the last three decades, there is a need for new, more efficient and less toxic compounds. Accordingly, the ability to identify such compounds is of paramount importance.